L-arabinose operon

{{short description|Gene regulation group}} The '''L-arabinose operon''', also called the '''''ara''''' or '''''araBAD'' operon''', is an operon required for the breakdown of the five-carbon sugar L-arabinose in ''Escherichia coli''.<ref name="Voet2011">{{cite book |last1=Voet |first1=Donald |url=https://archive.org/details/biochemistrythed00voet |title=Biochemistry |last2=Voet |first2=Judith G. |date=2011 |publisher=John Wiley & Sons |isbn=978-0470-57095-1 |edition=4th. |location=Hoboken, NJ |pages=[https://archive.org/details/biochemistrythed00voet/page/n1320 1291]–1294 |url-access=limited}}</ref> The L-arabinose operon contains three structural genes: ''araB'', ''araA'', ''araD'' (collectively known as ''araBAD''), which encode for three metabolic enzymes that are required for the metabolism of L-arabinose.<ref name="Schleif2000">{{cite journal|last1=Schleif|first1=Robert|title=Regulation of the L-arabinose operon of ''Escherichia coli''|journal=Trends in Genetics|volume=16|issue=12|year=2000|pages=559–565|doi=10.1016/S0168-9525(00)02153-3|pmid=11102706|doi-access=free}}</ref> AraB (ribulokinase), AraA (an isomerase), and AraD (an epimerase) produced by these genes catalyse conversion of L-arabinose to an intermediate of the pentose phosphate pathway, D-xylulose-5-phosphate.<ref name="Schleif2000"/>

The structural genes of the L-arabinose operon are transcribed from a common promoter into a single transcript, a mRNA.<ref name="Watson">{{cite book|last1=Watson|first1=James D. |title=Molecular biology of the gene|date=2008|publisher=Addison-Wesley|location=Harlow|isbn=9780321507815|pages=634–635|edition= 6th.}}</ref> The expression of the L-arabinose operon is controlled as a single unit by the product of regulatory gene ''araC'' and the catabolite activator protein (CAP)-cAMP complex.<ref name="Schleif2010">{{cite journal|last1=Schleif|first1=Robert|title=AraC protein, regulation of the l-arabinose operon in, and the light switch mechanism of AraC action|journal=FEMS Microbiology Reviews|volume=34|issue=5|year=2010|pages=779–796|doi=10.1111/j.1574-6976.2010.00226.x|pmid=20491933|doi-access=free}}</ref> The regulator protein AraC is sensitive to the level of arabinose and plays a dual role as both an activator in the presence of arabinose and a repressor in the absence of arabinose to regulate the expression of ''araBAD''.<ref>{{cite journal|last1=Lobell|first1=R. B.|last2=Schleif|first2=R. F.|title=DNA looping and unlooping by AraC protein.|journal=Science|date=1990|volume=250|issue=4980|pages=528–532|pmid=2237403|doi=10.1126/science.2237403|bibcode=1990Sci...250..528L|s2cid=25017204}}</ref> AraC protein not only controls the expression of ''araBAD'' but also auto-regulates its own expression at high AraC levels.<ref name="Schleif2003">{{cite journal|last1=Schleif|first1=Robert|title=AraC protein: A love-hate relationship|journal= BioEssays|year=2003|volume=25|issue=3|pages=274–282|doi=10.1002/bies.10237|pmid=12596232}}</ref>

==Structure== L-arabinose operon is composed of structural genes and regulatory regions including the operator region (''araO1'', ''araO2'') and the initiator region (''araI1'', ''araI2'').<ref name="Schleif1975">{{cite journal|last1=Schleif|first1=Robert|last2=Lis|first2=John T.|title=The regulatory region of the l-arabinose operon: A physical, genetic and physiological study|journal=Journal of Molecular Biology|date=1975|volume=95|issue=3|pages=417–431|doi=10.1016/0022-2836(75)90200-4|pmid=168391}}</ref> The structural genes, ''araB'', ''araA'' and ''araD'', encode enzymes for L-arabinose catabolism. There is also a CAP binding site where CAP-cAMP complex binds to and facilitates catabolite repression, and results in positive regulation of ''araBAD'' when the cell is starved of glucose.<ref name="Ogden1980">{{cite journal|last1=Ogden|first1=S|last2=Haggerty|first2=D|last3=Stoner|first3=CM|last4=Kolodrubetz|first4=D|last5=Schleif|first5=R|title=The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.|journal=Proceedings of the National Academy of Sciences of the United States of America|date=1980|volume=77|issue=6|pages=3346–3350|pmid=6251457|doi=10.1073/pnas.77.6.3346|pmc=349612|bibcode=1980PNAS...77.3346O|doi-access=free}}</ref> center|thumb|600x600px|Structure of L-arabinose operon of ''E. coli.'' The regulatory gene, ''araC'', is located upstream of the L-arabinose operon and encodes the arabinose-responsive regulatory protein AraC. Both ''araC'' and ''araBAD'' have a discrete promoter where RNA polymerase binds and initiates transcription.<ref name="Schleif2010"/> ''araBAD'' and ''araC'' are transcribed in opposite directions from the ''araBAD'' promoter (''PBAD'') and ''araC'' promoter (''PC'') respectively.<ref name="Schleif2000"/>

==Function==

*''araA'' encodes L-arabinose isomerase, which catalyses isomerization between L-arabinose and L-ribulose. *''araB'' encodes ribulokinase, which catalyses phosphorylation of L-ribulose to form L-ribulose-5-phosphate. *''araD'' encodes L-ribulose-5-phosphate 4-epimerase, which catalyses epimerization between L-ribulose 5-phosphate and D-xylulose-5-phosphate. 600px|thumb|left|Metabolic pathway of L-arabinose via the action of three enzymes, which are encoded by the ''araBAD'' operon. {| class="wikitable" |+ Catabolism of arabinose in ''E. coli'' ! Substrate !! Enzyme(s) !! Function !! Reversible !! Product |- |{{small|L}}-arabinose || AraA || Isomerase || Yes || {{small|L}}-ribulose |- |{{small|L}}-ribulose || AraB || Ribulokinase || No || {{small|L}}-ribulose-5-phosphate |- |{{small|L}}-ribulose-5-phosphate || AraD || Epimerase || Yes || {{small|D}}-xylulose-5-phosphate |}

Both L-ribulose 5-phosphate and D-xylulose-5-phosphate are metabolites of the pentose phosphate pathway, which links the metabolism of 5-carbon sugars to that of 6-carbon sugars.<ref name="Schleif2003"/>

==Regulation==

250px|thumb|right|Structure of AraC monomer The L-arabinose system is not only under the control of CAP-cAMP activator, but also positively or negatively regulated through binding of AraC protein. AraC functions as a homodimer, which can control transcription of ''araBAD'' through interaction with the operator and the initiator region on L-arabinose operon. Each AraC monomer is composed of two domains including a DNA binding domain and a dimerisation domain.<ref name="Bustos1993">{{cite journal|last1=Bustos|first1=S. A|last2=Schleif|first2=R. F|title=Functional domains of the AraC protein.|journal=Proceedings of the National Academy of Sciences of the United States of America|date=1993|volume=90|issue=12|pages=5638–5642|pmid=8516313|doi=10.1073/pnas.90.12.5638|pmc=46776|bibcode=1993PNAS...90.5638B|doi-access=free}}</ref> The dimerisation domain is responsible for arabinose-binding.<ref name="Saviola1998">{{cite journal|last1=Saviola|first1=B|last2=Seabold|first2=R|last3=Schleif|first3=R. F|title=Arm-domain interactions in AraC.|journal=Journal of Molecular Biology|date=1998|volume=278|issue=3|pages=539–548|doi=10.1006/jmbi.1998.1712|pmid=9600837|doi-access=free}}</ref> AraC undergoes conformational change upon arabinose-binding, in which, it has two distinct conformations.<ref name="Schleif2003"/> The conformation is purely determined by the binding of allosteric inducer arabinose.<ref name="Griffiths">{{cite book|last1=Griffiths|first1= Anthony J.|last2=Wessler|first2= Susan R.|title=Introduction to genetic analysis|date=2015|publisher=Freeman|location=New York, NY|isbn=9781429276344|pages=413–414|edition= 11th}}</ref>

AraC can also negatively autoregulate its own expression when the concentration of AraC becomes too high. AraC synthesis is repressed through binding of dimeric AraC to the operator region (''araO1'').

===Negative regulation of ''araBAD''=== 500px|thumb|right|Negative regulation of L-arabinose operon via AraC protein When arabinose is absent, cells do not need the ''araBAD'' products for breaking down arabinose. Therefore, dimeric AraC acts as a repressor: one monomer binds to the operator of the ''araBAD'' gene (''araO2''), another monomer binds to a distant DNA half site known as ''araI1''.<ref name="Casadaban">{{cite journal|last1=Casadaban|first1=Malcolm J.|title=Regulation of the regulatory gene for the arabinose pathway, ''araC''|journal=Journal of Molecular Biology|date=1976|volume=104|issue=3|pages=557–566|doi=10.1016/0022-2836(76)90120-0|pmid=781294}}</ref> This leads to the formation of a DNA loop.<ref name="Seabold1998">{{cite journal|last1=Seabold|first1=Robert R|last2=Schleif|first2=Robert F|title=Apo-AraC actively seeks to loop|journal=Journal of Molecular Biology|year=1998|volume=278|issue=3|pages=529–538|doi=10.1006/jmbi.1998.1713|pmid=9600836|doi-access=free}}</ref> This orientation blocks RNA polymerase from binding to the ''araBAD'' promoter.<ref name="Hendrickson1984">{{cite journal|last1=Hendrickson|first1=William|last2=Schleif|first2=Robert|title=Regulation of the ''Escherichia coli'' L-arabinose operon studied by gel electrophoresis DNA binding assay|journal=Journal of Molecular Biology|date=1984|volume=178|issue=3|pages=611–628|doi=10.1016/0022-2836(84)90241-9|pmid=6387154}}</ref> Therefore, transcription of structural gene ''araBAD'' is inhibited.<ref name="Weaver2012">{{cite book|last1=Weaver|first1=Robert Franklin|title=Molecular biology|url=https://archive.org/details/molecularbiology00weav_632|url-access=limited|date=2012|publisher=McGraw-Hill|location=New York|isbn=9780071316866|pages=[https://archive.org/details/molecularbiology00weav_632/page/n205 183]–186|edition= 5th int. student}}</ref>

===Positive regulation of ''araBAD''=== 500px|thumb|right|Positive regulation of L-arabinose operon via dimeric AraC and CAP/cAMP Expression of the ''araBAD'' operon is activated in the absence of glucose and in the presence of arabinose. When arabinose is present, both AraC and CAP work together and function as activators.<ref name="Snyder">{{cite book|last1=Snyder|first1=Larry|title=Molecular genetics of bacteria|date=2013|publisher=ASM Press|location=Washington, DC|isbn=9781555816278|pages=487–494|edition= 4th.}}</ref>

====Via AraC==== AraC acts as an activator in the presence of arabinose. AraC undergoes a conformational change when arabinose binds to the dimerization domain of AraC. As a result, the AraC-arabinose complex falls off from ''araO2'' and breaks the DNA loop. Hence, it is more energetically favourable for AraC-arabinose to bind to two adjacent DNA half sites: ''araI1'' and ''araI2'' in the presence of arabinose. One of the monomers binds ''araI1'', the remaining monomer binds ''araI2'' - in other words, binding of AraC to ''araI2'' is allosterically induced by arabinose. One of the AraC monomers places near to the ''araBAD'' promoter in this configuration, which helps to recruit RNA polymerase to the promoter to initiate transcription.<ref name="Hartnell">{{cite book|last1=Hartwell|first1=Leland|last2=Hood|first2=Leroy|title=Genetics : from genes to genomes|date=2010|publisher=McGraw-Hill Education|location=Boston|isbn=9780071102155|page=[https://archive.org/details/genetics00lela_0/page/528 528]|edition=4th|url=https://archive.org/details/genetics00lela_0/page/528}}</ref>

====Via CAP/cAMP (catabolite repression)====

CAP act as a transcriptional activator only in the absence of ''E. coli'''s preferred sugar, glucose.<ref name="Cox">{{cite book|last1=Cox|first1=Michael M. |last2=Doudna|first2=Jennifer A.|last3=O'Donnell|first3=Michael E.|title=Molecular biology : principles and practice|date=2012|publisher=W.H. Freeman|location=New York|isbn=9781464102257|pages=707–708|edition= International}}</ref> When glucose is absent, high level of CAP protein/cAMP complex bind to CAP binding site, a site between ''araI1'' and ''araO1''.<ref name="Griffiths2002">{{cite book|last1=Griffiths|first1=Anthony J.F.|title=Modern genetic analysis: integrating genes and genomes|date=2002|publisher=W.H. Freeman|location=New York|isbn=0716743825|pages=[https://archive.org/details/lccn_2001059225/page/432 432–433]|edition=2nd.|url-access=registration|url=https://archive.org/details/lccn_2001059225/page/432}}</ref> Binding of CAP/cAMP is responsible for opening up the DNA loop between ''araI1'' and ''araO2'', increasing the binding affinity of AraC protein for ''araI2'' and thereby promoting RNA polymerase to bind to ''araBAD'' promoter to switch on the expression of the ''araBAD'' required for metabolising L-arabinose.

500px|thumb|right|Autoregulation of ''araC'' expression

===Autoregulation of AraC===

The expression of ''araC'' is negatively regulated by its own protein product, AraC. The excess AraC binds to the operator of the ''araC'' gene, ''araO1'', at high AraC levels, which physically blocks the RNA polymerase from accessing the ''araC'' promoter.<ref name="Lee1981">{{cite journal|last1=Lee|first1=N. L|last2=Gielow|first2=W.O|last3=Wallace|first3=R. G|title=Mechanism of ''araC'' autoregulation and the domains of two overlapping promoters, ''Pc'' and ''PBAD'', in the L-arabinose regulatory region of ''Escherichia coli''.|journal=Proceedings of the National Academy of Sciences of the United States of America|year= 1981|volume=78|issue=2|pages=752–756|pmid=6262769|doi=10.1073/pnas.78.2.752|pmc=319880|bibcode=1981PNAS...78..752L|doi-access=free}}</ref> Therefore, the AraC protein inhibits its own expression at high concentrations.<ref name="Snyder"/>

==Use in protein expression system==

The L-arabinose operon has been a focus for research in molecular biology since 1970, and has been investigated extensively at its genetic, biochemical, physiological and biotechnical levels.<ref name="Watson"/> The L-arabinose operon has been commonly used in protein expression system, as the ''araBAD'' promoter can be used for producing targeted expression under tight regulation. By fusing the ''araBAD'' promoter to a gene of interest, the expression of the target gene can be solely regulated by arabinose: for example, the pGLO plasmid contains a green fluorescent protein gene under the control of the ''PBAD'' promoter, allowing GFP production to be induced by arabinose.

==See also== *Operon *Catabolism *Catabolite repression Other operon systems in ''E. coli'': *''gal'' operon *''gab'' operon *''lac'' operon *''trp'' operon

==References== {{Reflist}}

==External links== *[https://www.ncbi.nlm.nih.gov/books/NBK21277/ Modern Genetic Analysis]{{dead link|date=July 2025|bot=medic}}{{cbignore|bot=medic}} by Griffiths, A.J et al. (online textbook) *[https://www.ncbi.nlm.nih.gov/books/NBK22512/ Biochemistry]{{dead link|date=July 2025|bot=medic}}{{cbignore|bot=medic}} by Berg, J.M et al. (online textbook) *[https://www.ncbi.nlm.nih.gov/books/NBK21849/ An Introduction to Genetic Analysis]{{dead link|date=July 2025|bot=medic}}{{cbignore|bot=medic}} by Griffiths, A.J et al. (online textbook)

Category:Gene expression Category:Operons